Conditional depletion of Smad1 and Smad5 reveals differing contributions of each to primitive hematopoiesis. Cre/loxP-mediated homologous recombination was used to target insertion of constructs containing shRNA hairpins corresponding to sequences within the MH1 domain of Smad1 alone (miSMAD1), Smad5 alone (miSMAD5), or both Smad5 and Smad1 (miSMAD51). Each hairpin construct is targeted to the identical site downstream of an operator/promoter, which allows tet-on activation via dox treatment (miSMAD1 was previously characterized³³ ). (A) Representative western blotting analysis of SMAD5 and SMAD1 protein expression in lysates derived from EBs after 24 hours of dox treatment (DOX, compared with uninduced control). (B) Quantitative densitometric analysis of samples induced with 2 μg/mL dox normalized to control β-actin levels from at least 3 separate experiments to derive mean ± SEM. (C-E) Embryoid bodies derived from parental AinV18 cells, miSMAD5, miSMAD51, or miSMAD1 ESCs were induced with dox on day 2 (dox d2) or day 4 (dox d4) of EB differentiation and evaluated by hematopoietic colony assays to measure primitive erythroid (EryP [C]) and myeloid (macrophages, MacP [D]; megakaryocytes, MegaP [E]) potential. Colony assays were each performed at least 3 separate times, with samples counted in triplicate, and values are reported as normalized to untreated controls. Statistical significance compared with no dox controls, with *P < .05 and **P < .01, respectively. CTL, control; dox, doxycycline.